Effect of heparin on the reactivity of SH-groups in the macrophage membrane

The effect of heparin on macrophage (M phi) adherence and on the reactivity of membrane SH-groups to the specific SH-oxidizing agent 4,4'-dithiodipyridine (PDS) was studied. Various types of SH-reactive agents, except 5,5'-dithio-bis (2-nitrobenzoate) (DTNB), were found to inhibit adherence of mouse peritoneal M phi to serum-coated Falcon surfaces. Heparin inhibited M phi-adherence in serum containing medium and in higher concentrations stimulated the adherence inhibitory effect of PDS, especially in Ca-depleted medium. This effect of heparin may be due to its polyanionic character, as dextran sulphate but not dextran induced similar changes. The effect of heparin to increase the sensitivity of membrane SH-groups against SH-reactive agents was demonstrated also by cytotoxicity experiments. It is concluded that heparin makes the M phi-membrane unstable, by exposing some hidden SH-groups playing a role in membrane function.     

Effects of anticoagulants upon sister-chromatid exchanges, cell-cycle kinetics, and mitotic index in human peripheral lymphocytes

The effects of 3 blood anticoagulants, heparin, acid citrate dextrose (ACD), and ethylenediaminetetraacetic acid (EDTA) were investigated using human peripheral lymphocytes. Three different endpoints were examined: sister-chromatid exchange (SCE), cell kinetics index (CKI), and mitotic index (MI). SCEs were significantly increased in cells treated with EDTA, while the CKI and MI were significantly decreased in cultures treated with either ACD or EDTA when compared to cultures treated with heparin. These results suggest that anticoagulants may produce undesired effects upon cultured cells and indicate that the type of anticoagulant should be considered carefully prior to commencing cytogenetic studies using human peripheral lymphocytes.     

Lymphocytosis produced by heparin and other sulphated polysaccharides in mice and rats

Lymphocytosis has been produced in mice and rats using heparin and other sulphated polysaccharides. Two hours after heparin (50 mg/kg ip) the concentration of lymphocytes in mouse blood increased threefold; it fell to control levels after 9 hr. The height of the lymphocytosis was related to the dose of heparin injected. After intravenous heparin in rats there was a comparable lymphocytosis maximal 1 hr after injection. In mice other negatively charged sulphated polysaccharides also caused lymphocytoses, which were greater and occurred later with increase in molecular weight of the substance injected. Results in rats were similar. No lymphocytosis followed the injection of negatively charged phosphated dextrans, positively charged DEAE dextran, or neutral dextran. There was no correlation between the effect of these substances on lymphocytes and their effect on coagulation of blood, hepatic phagocytosis, or the immune response to sheep red blood cells.     

Complexes of glucose oxidase with chitosan and dextran possessing enhanced stability

Abstract

In this study, the different mole ratios of glucose oxidase/chitosan/dextran–aldehyde and glucose oxidase/chitosan/dextran–sulfate complexes were synthesized. The modification of glucose oxidase by non-covalent complexation with dextran and chitosan in different molar ratios was studied in order to increase the enzyme activity. The enzyme/polymer complexes obtained were investigated by UV spectrophotometer and dynamic light scattering. Activity determination of synthesized complexes and free enzyme were performed at a temperature range. The best results were obtained by C_chitosan/C_{dextran–aldehyde} = 10/1 ratio and C_chitosan/C_{dextran–sulfate} = 1/5 ratio that were used in thermal stability, shelf life, salt stress, and ethanol effect experiments. The results demonstrated that both complexes were thermally stable at 60?°C and had superior storage stability compared to the free glucose oxidase. Complexes showed higher enzymatic activity than free enzyme in the organic solvent environment using 10% ethanol. The complexes were resistant to salt stress containing 0.1?M NaCl or CaCl₂. The particle size distribution results of the triple complex evaluated the complexation of the chitosan, dextran derivative, and glucose oxidase. The average size of the triple complex in diameter was found to be 325.8?±?9.3?nm. Overall findings suggest that the complexes of glucose oxidase, chitosan, and dextran showed significant enhancement in the enzyme activity.     

Improved functional recovery of human granulocytes after cryopreservation

Human peripheral blood phagocytes (90% neutrophils) were cryopreserved with either 5 or 10% dimethyl sulfoxide (DMSO) and stored in the liquid phase of liquid nitrogen. Modifications to the freezing method included the elimination of dextran from the freezing medium, addition of the bulk of the DMSO at −5 °C, elimination of heparin and centrifugation from all postreconstitution procedures, and the use of deoxyribonuclease to minimize post-thaw granulocyte agglutination.Substantial numbers of the cryopreserved phagocytes, as assayed by nitroblue tetrazolium and chemotactic activity, showed comparable functional activity to fresh cells. Post-thaw cell dialysis further improved functional capacity although probably not as a consequence of DMSO removal.     

Factors influencing human leukocyte adherence in vivo

The effects of different factors on leukocyte adherence was evaluated by using human peripheral blood before and after treatment (application). Magnesium ions, anti-inflammatory drugs and dextran have decreased neutrophil adherence. Calcium ions, propranolol, blood transfusion caused a significant decrease in adherence. These findings support the conclusion that the adherence test, which is influenced by numerous factors and whose results had a wide scatter, is not a useful tool in clinical diagnosis.     

Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa

The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P <0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.     

Incidences of fatal postoperative pulmonary embolism after prophylaxis with dextran 70 and low-dose heparin: an international multicentre study.

A total of 4352 patients were admitted to a prospective'' randomised multicentre trial comparing the prophylactic efficacy of dextran 70 and low-dose heparin against fatal pulmonary embolism after elective operations for general, orthopaedic, urological, and gynaecological conditions. Out of 3984 patients correctly admitted, 1993 were allocated to receive dextran 70 and 1991 to receive low-dose heparin. Withdrawal of prophylaxis because of bleeding or technical difficulties occurred more often in the heparin group, but allergic reactions were more common in the dextran group. Of the 75 patients who died within 30 days after operation, 38 had been given dextran and 37 low-dose heparin. Necropsy was performed in 33 and 32 of these cases respectively. In six patients in each group pulmonary embolism was the sole or a contributory cause of death. Of these, five patients in the dextran group and two in the heparin group had received a full course of prophylaxis. There was no statistically significant difference between the two treatment groups in the incidence of fatal pulmonary embolism after a full course of prophylaxis.     

Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate, and its mechanism.

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VIII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content of dextran sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.     

Proteomic analysis of Saccharomyces cerevisiae under high gravity fermentation conditions

Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g/L (high). Although grown in a high glucose environment, S. cerevisiae still retained the ability to produce ethanol with a high degree of glucose utilization. iTRAQ-mediated shotgun proteomics was applied to identify relative expression change of proteins under the different glucose conditions. A total of 413 proteins were identified from three replicate, independent LC-MS/MS runs. Unsurprisingly, many proteins in the glycolysis/gluconeogenesis pathway showed significant changes in expression level. Twenty five proteins involved in amino acid metabolism decreased their expression, while the expressions of 12 heat-shock related proteins were also identified. Under high glucose conditions, ethanol was produced as a major product. However, the assimilation of glucose as well as a number of byproducts was also enhanced. Therefore, to optimize the ethanol production under very high gravity conditions, a number of pathways will need to be deactivated, while still maintaining the correct cellular redox or osmotic state. Proteomics is demonstrated here as a tool to aid in this forward metabolic engineering.     

Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate,and its mechanism

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.     

Recovery of functional capacity of human granulocytes after freeze-preservation with dimethyl sulphoxide.

Huma peripheral blood leucocytes (neutrophil rich) were collected either with preservative-free heparin (PFH) or acid citrate dextrose (ACD), frozen with dimethyl sulphoxide (DMSO) at a controlled rate, stored in liquid nitrogen at ?196 °C and reconstituted in a solution containing dextran polymer 70.A battery of tests including nitroblue tetrazolium (NBT) reduction, Candida phagocytic and candidacidal capacity was used to compare anticoagulants and reconstitution methods as they affect functional capacity of freeze-thawed neutrophils during a short post-thaw period.Heparin showed an overall advantage over acid citrate dextrose. A slow titration reconstitution method did not improve cell yield or functional capacity compared with a rapid dilution method and was a more cumbersome technique. The presence of complement greatly improved the capacity of reconstituted cells to reduct NBT and synthesize formazan. Freeze-thawed cells showed a selective response to stimulation as judged by the quantitative NBT test, responding strongly to zymosan in comparison with E. coli endotoxin. Lignocaine hydrochloride added to the reconstituent medium in concentrations up to 20 mmol/l did not have additional protective effect on post-thawed leucocytes as assessed by agglutination and leucocyte yields when compared with reconstitutent solutions containing only dextran. Reducing pH did not significantly slow the rate of gelling and leuco-agglutination or improve cell yields.Using these findings to optimize conditions reconstituted neutrophils retained 31.7% of fresh NBT activity, 27.6% of fresh phagocytic, and 22.3% of fresh candidacidal capacity.     

Direct observation of fibrinogen-heparinoid complexes formation using surface plasmon resonance.

We analyzed the binding of heparinoid or heparin with fibrinogen by real-time measurement using surface plasmon resonance technology. Poly(glucosyloxyethyl methacrylate) sulfate [poly(GEMA) sulfate] and dextran sulfate were used as heparinoids. The binding ability of each sulfated polymer was estimated by having each polymer-containing buffer interact with the sensor chip surfaces that had immobilized fibrinogen. Dextran sulfate and poly(GEMA) sulfate showed high affinity to the fibrinogen in this experiment, while the heparin did not. All of the dextran sulfates were desorbed from its surface, while about 30% of the poly(GEMA) sulfate remained on the immobilized fibrinogen upon the addition of NaCl to the buffer which was done in order to analyze the desorption of poly(GEMA) sulfate or dextran sulfate from the surface of the fibrinogen. These data show that the type of binding between fibrinogen-poly(GEMA) sulfate was different from that of dextran sulfate, indicating that the interaction between fibrinogen and poly(GEMA) sulfate was caused not only by an electrostatic but also by a hydrophobic force. These results suggest that the interaction mechanism of heparinoids with fibrinogen was different from that of heparin.     

Gender-specific response to interstitial angiotensin II in human white adipose tissue.

Angiotensin II is synthesized locally in various tissues. However, the role of interstitial angiotensin II in the regulation of regional metabolism and tissue perfusion has not as yet been clearly defined. We characterized the effect of interstially applied angiotensin II in abdominal subcutaneous adipose tissue of young, normal-weight, healthy men (n = 8) and women (n = 6) using the microdialysis technique. Adipose tissue was perfused with 0.01, 0.1, and 1 micro M angiotensin II. Dialysate concentrations of ethanol, glycerol, glucose, and lactate were measured to assess changes in blood flow (ethanol dilution technique), lipolysis, and glycolysis, respectively. Baseline ethanol ratio and dialysate lactate were both significantly higher, whereas dialysate glucose was significantly lower in men vs. women. In men, ethanol ratio and dialysate glucose, lactate and glycerol did not change significantly during perfusion with angiotensin II. In women, however, angiotensin II induced a significant increase in ethanol ratio and dialysate lactate and a decrease in dialysate glucose close to values found for men and this response was almost maximal at the lowest angiotensin II concentration used. Dialysate glycerol did not change significantly. We conclude that baseline blood flow and glucose supply and metabolism is significantly higher in women than in men. In men, interstitial Ang II has only a minimal effect on adipose tissue blood flow and metabolism. In women, however, a high physiological concentration of interstitial angiotensin II can reduce blood flow down to values found in men. This is associated with an impaired glucose supply and metabolism. Additionally, Ang II inhibits lipolysis.     

Effect of ultraviolet irradiation of autologous blood on cell volumes, cell adhesion and phagocytosis in normal probands and patients with multiple sclerosis

UVB induced changes of blood cell properties were investigated in 12 MS patients and in 10 healthy volunteers serving as normal controls. The mean cell volume (MCV) was determined by electronic sizing, the granulocyte and lymphocyte adherence was estimated in a capillary assay, and the phagocytic activity of granulocytes was measured in a test system based on the incorporation of opsonized baker's yeast (Saccharomyces cerevisiae). In MS patients the MCV of red cells and lymphocytes decreased rapidly within 6 UVB treatments. In contrast, the reduction of the granulocyte volume was delayed (between the 6th and 12th UVB). In the control group the mean value of the red cell and lymphocyte MCV remained rather unaffected. There was a slight rise of the granulocyte volume after the 6th UVB. The only significant change of adherence was an increase of granulocyte adherence in MS patients. Untreated patients had a significantly enhanced phagocytic activity in comparison to the control group. 6 UVB treatments included a significant reduction of the phagocytic activity in MS patients. However, subsequently the percentage of phagocytizing cells increased again, whereas the particle uptake per cell continued to decrease. In the control group only minor UVB induced changes of phagocytosis were observed. The in vitro UV irradiation caused an enhanced phagocytosis in the majority of cases in both controls and MS patients. In general, under the UVB treatment all parameters examined changed in the sense of a normalisation, in that the measured values reached a new level lying between the extreme pretreatment values accompanied by a reduced standard deviation. The effect of UVB was more pronounced in MS patients when compared with normal controls. This could result from an enhanced sensitivity to the influence of UVB of pathologically altered cells in MS patients. The monitoring of the MCV of red cells and lymphocytes as well as the repeated testing of granulocyte phagocytosis are recommended for supportion of therapy planning and follow-up of MS patients.     

Studies on the interaction of leucocytes and the myocardial vasculature

Granulytes play an important role in increasing the infarct size after ischemia and reperfusion by the release of oxygen-derived free radicals (ODFR) and lysosomal enzymes. It has been shown that the number of granulocytes adhering to the vascular endothelium increases after occlusion of the coronary artery, and that the area of myocardial damage can be reduced by preventing granulocyte adherence with monoclonal antibodies directed against adhesion receptors. The underlying mechanism of granulocyte activation under these conditions is not yet known. We have investigated whether granulocytes can be activated directly by reduced oxygen tensions. Granulocytes were suspended in a hypoxic buffer and incubated on fibronectin and gelatin coated microtitre plates at 1–3% ambient oxygen to study their ability to adhere to these matrices. The results showed that the adherence of granulocytes to fibronectin was dependent on the duration of hypoxia. After 30 min of incubation under hypoxia granulocyte adherence increased 1.3 to 1.8 fold compared to the normoxic control. The adherence to fibronectin could be inhibited partially by anti-CD18 antibody, a monoclonal antibody to the common beta chain of a class of extracellular matrix receptors. This direct activation of granulocytes due to hypoxic conditions may have implications for the interaction of these cells with the vascular endotheliumin vivo, (Mol Cell Biochem116: 197–202, 1992)     

Low molecular weight heparin in prevention of perioperative thrombosis.

OBJECTIVE--To determine whether prophylactic treatment with low molecular weight heparin reduces the incidence of thrombosis in patients who have had general or orthopaedic surgery. DESIGN--Meta-analysis of results from 52 randomised, controlled clinical studies (29 in general surgery and 23 in orthopaedic surgery) in which low molecular weight heparin was compared with placebo, dextran, or unfractionated heparin. SUBJECTS--Patients who had had general or orthopaedic surgery. INTERVENTION--Once daily injection of a low molecular weight heparin compared with placebo, dextran, or unfractionated heparin. MAIN OUTCOME MEASURES--Incidence of deep venous thrombosis, pulmonary embolism, major haemorrhages, and death. RESULTS--The results confirm that low molecular weight heparins are more efficacious for the prophylactic treatment of deep venous thrombosis than placebo (common odds ratio 0.31, 95% confidence interval 0.22 to 0.43; p < 0.001) and dextran (0.44, 0.30 to 0.65; p < 0.001). The results suggest that low molecular weight heparins are also more efficacious than unfractionated heparin (0.85, 0.74 to 0.97; p = 0.02), with no significant difference in the incidence of major haemorrhages (1.06, 0.93 to 1.20; p = 0.62). CONCLUSIONS--Low molecular weight heparins seem to have a higher benefit to risk ratio than unfractionated heparin in preventing perioperative thrombosis. However, it remains to be shown in a suitably powered clinical trial whether low molecular weight heparin reduces the risk of fatal pulmonary embolism compared with heparin.     

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells (P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.     

Parameters affecting the yield of DNA from human blood

An examination of variables affecting the yield of DNA from blood was undertaken in order to improve sample processing and to evaluate alternative methods of mailing blood samples for DNA analysis. A rapid, high-yield method was developed for the isolation of high-molecular-weight DNA from fresh and frozen blood. In addition, the following observations were made: (1) Of the anticoagulants examined, acid citrate dextrose (ACD) solution B was found to be superior to EDTA and heparin for preserving yields of DNA during incubation at room temperature. If DNA is isolated from frozen blood, high yields of undegraded DNA are achieved after incubation at 23 degrees C for 5 days with ACD solution B. (2) High yields of undegraded DNA are obtained from blood stored with ACD solution B for at least 1 day at 42 degrees C, 5 days at 0 degrees C, or 1 month at -20 degrees C. (3) Three cycles of freezing and thawing may have little if any affect on the yield of DNA. The results indicate that blood for DNA extraction may be mailed in an ambient temperature container and, in many cases, sent by first-class mail rather than by overnight delivery services.     

Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations.

We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward-angle scatter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque-Ficoll (HF) separation and four whole blood (WB) lysis techniques: Immuno-lyse, Q-Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lower values. Autofluorescence values were comparable by all methods except Immuno-lyse, which showed consistently higher values in blood stored for 24 h with any anticoagulant. Immunofluorescent values for CD45-stained cells were quite consistent across all methods, and among the whole blood methods, FACS Lyse and Q-Prep uniformly gave the highest purity of CD45-positive cells in the lymphocyte light scatter gates. Additionally, propidium iodide (PI) analyses of CD45-stained whole blood, and analyzed without lysis, confirmed that ACD and heparin were superior to EDTA for maintaining viable leucocytes overnight. Future studies should focus on other commonly used reagents, a wide variety of abnormal samples, and cell viability.